Expression of equine morbillivirus (EMV) matrix and fusion proteins and their evaluation as diagnostic reagents.

This scientific piece focuses on the creation of cDNA clones for the matrix and fusion proteins of the equine morbillivirus (EMV) to assess their effectiveness as diagnostic reagents. The proteins were converted using two different systems and their stability and antigenic properties were evaluated. An affinity-purified truncated protein demonstrated promising application in diagnosing EMV infection through Western blot and ELISA-based screening.

Procedure for Cloning EMV Proteins

• The researchers started by creating complete cDNA clones coding for the matrix (M) and fusion (F) proteins of the equine morbillivirus (EMV). This process was achieved through reverse transcription-polymerase chain reaction (RT-PCR), a method that permits the production of many copies of the genetic material being studied.
• These copies were then expressed in Escherichia coli , a common bacterium used in genetic experiments, using two different expression systems.

Protein Expression and Analysis

• Western blot analysis, a technique used to detect specific proteins, indicated that both the M and F proteins, whether expressed individually or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression.
• Further analysis of the degradation pattern of the recombinant M protein suggested that the N-terminus (the start of the protein chain) of the matrix protein might be more stable and produce more of an immune response (be more antigenic) than the C-terminal region.

Creation of Stable Truncated Protein

• To address this, a third expression system was used to express a truncated version of the M protein. This truncated version contained only the N-terminal amino acid residues 1-197, with a (His)6-tag added at the start of the protein.
• Although this new recombinant protein, known as (His)6-Mtr, was found to be stable, it was also insoluble.

Evaluation and Diagnostic Application

• After a one-step affinity purification under denaturing conditions, (His)6-Mtr was used to monitor the antibody response to EMV infection through a Western blot analysis and enzyme-linked immunosorbent assay (ELISA).
• The researchers found a 100% correlation between the Western blot results and virus neutralization testing, suggesting that the recombinant protein can effectively identify EMV infections. However, they also noted that the number of positive samples available for testing was minimal, limiting the robustness of these initial results.